1. Field of the Invention
The present invention relates to a gene (isolated nucleic acid molecule) encoding the cyclodextrin glucanotransferase which produces a considerable amount of γ-cyclodextrin (γ-CD) from the substrates selected from among starch and starch decomposition products such as dextrin, amylopectin and amylose; recombinant plasmids comprising this gene; transformants transformed with the recombinant plasmid; methods of manufacturing the cyclodextrin glucanotransferase by employing these transformants to act upon the substrates selected from among starch and decomposition products thereof and causing the production of γ-CD as a main product; and methods of manufacturing γ-CD and CD-comprising compositions having a desired CD balance (α-, β- and γ-CD balance) employing this recombinant cyclodextrin glucanotransferase.
2. Description of Related Art
Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) is an enzyme that acts on α-1,4-glucans such as starch and produces cyclodextrins (CDs), a cyclic α-1,4-glucan, by intermolecular transglycosylation activity. CDs composed of 6, 7 and 8 D-glucosyl moieties are called as α-, β- and γ-CD respectively. Aside from this CD-producing reaction, through intermolecular transglycosylation, CGTase also catalyzes coupling reactions (reactions in which the CD ring is opened and the straight-chain oligosaccharides produced are transferred to receptor sugar molecules) and disproportionation reactions (in which straight-chain oligosaccharides are transferred to receptor sugar molecules). Further, CGTase also weakly catalyzes hydrolysis of α-1,4-glucoside bonds. These CDs are interesting molecules from the viewpoints of food and medical applications and so on, because of their ability to from inclusion complexes with many organic and inorganic molecules, thereby changing the physical and chemical properties. (E. B. Tilden and S. J. Pirt, J. Am. Chem. Soc., 63, 2900-2902, 1939). Significant research has been conducted, including the search for CGTase-producing bacteria and the purification of the enzyme since the synthesis of CDs by the CGTase from Bacillus macerans was reported in 1939 (Sumio Kitahata, Naoto Tsuyama and Shigetaka Okada, Agr. Biol. Chem., 38 (2), 387-393, 1974; Sumio Kitahata and Shigetaka Okada, Agr. Biol. Chem., 38 (12), 2413-2417, 1974; Sumio Kitahata and Shigetaka Okada, J. Jap. Soc. Starch Sci., 29 (1), 13-18, 1982; Michio Kubota, Yoshiki Matsuura, Shuzo Sakai and Yukiteru Katsube, Denpun Kagaku, 38 (2), 141-146, 1991; Lionel J. Bovetto, Daniel P. Backer, Jaques R. Villette, Philippe J. Sicard, and Stephane J-L. Bouquelet, Biotechnology and Applied Biochemistry, 15, 48-58, 1992; Shinske Fujiwara, Hirofumi Kakihara, Kim Myung Woo, Andre Lejeune, Mitsuhide Kanemoto, Keiji Sakaguchi, and Tadayuki Imanaka, Applied and environmental microbiology, 58 (12), 4016-4025, 1992; Florian Binder, Otto Huber and August Bock, Gene, 47, 269-277, 1986; Keiji Kainuma, Toshiya Takano and Kunio Yamane, Appl. Microbiol. Biotechnol., 26, 149-153, 1987; Takahiro Kaneko, Tetsuo Hamamoto and Koki Horikoshi, J. general Microbiology, 134, 97-105, 1988; Murai Makela, Pekka Mattsson, M. Eugenia Schinina, and Timo Korpela, Biotechnology and Applied biochemistry, 10, 414-427, 1988; and Ernest K. C. Yu, Hiroyuki Aoki, and Masanaru Misawa, Appl. Microbiol. Biotechnol., 28, 377-379, 1988).
CGTases are classified into α-, β- and γ-CGTase depending on the main products of CD. Most reports have dealt with α- and β-CGTase; there are few enzymes reported to be γ-CGTase (Shigeharu Mori, Susumu Hirose, Takaichi Oya, and Sumio Kitahata, Oyo Toshitsu Kagaku, 41 (2), 245-253, 1994; Yoshito Fujita, Hitoshi Tsubouchi, Yukio Inagi, Keiji Tomita, Akira Ozaki, and Kazuhiro Nakanishi, J. Fermentation and Bioengineering, 70 (3), 150-154, 1990; Takashi Kato and Koki Horikoshi, J. Jpn. Soc. Starch Sci., 33 (2), 137-143, 1986). Further, of those enzymes reported as γ-CGTase, the γ-CD production yield is less than 5 percent; an amount of β-CD that is equal to or greater than the amount of γ-CD is produced since the rate of production of β-CD is accelerated in the late stage of reaction; or the γ-CD production rate decreases sharply at a substrate concentration of 10 percent or more. Still further, these enzymes do not lend themselves to an industrial use since they requires countermeasures such as the addition of ethanol to the reaction solution.
Some attempts to improve the amount of γ-CD produced by modifying the structural gene of α- or β-CGTase have been reported (Akira Nakamura, Keiko Haga, and Kunio Yamane, Biochemistry, 32, 6624-6631, 1993; Michio Kubota, Yoshiki Matsuura, Shuzo Sakai and Yukiteru Kutsume, Oyo Toshitsu Kagaku, 41 (2), 245-253, 1994). However, even when the amount of γ-CD produced increases, the amount of β-CD produced by the original activity does not markedly decrease, which is inadequate from an industrial perspective. As a result, although α-CD and β-CD are employed in various fields, γ-CD is hardly employed at all. The same is true of CD-comprising compositions. CD-comprising compositions containing α- or β-CD as a main product are employed in a great many fields, but CD-comprising compositions containing γ-CD as a main product are hardly employed at all. In CD-comprising compositions, the CGTase employed to prepare the compositions ends up determining the CD composition based on α-, β-, or γ-CGTase, and it is difficult to prepare a CD-comprising composition having a desired CD balance.
In light of this state of the art, the present inventors previously discovered that Bacillus clarkii 7364 produces a new γ-CGTase producing γ-CD as a main product, employed this enzyme to develop a method of manufacturing γ-CD and a CD-comprising composition of desired CD balance, and applied for patents (Japanese Patent Application Un-examined Publication Nos. 2001-327299 and 2001-327284).
However, these microbes do not afford adequate enzyme productivity. When γ-CD and CD-comprising compositions of desired CD balance are prepared on an industrial scale, there is a problem in that the microbe must be cultured on a large scale. Conventionally, to solve this problem, wild strains have been bred in a complex manner using ultraviolet radiation, X-rays, and reagents such as NTG (N-methyl-N′-nitro-N-nitrosoguanidine) and EMS (ethylmethane sulfonate) to create mutant strains having improved enzyme productivity. Further, microbes producing CGTase often produce trace amounts of α-amylase at the same time as CGTase. Thus, when causing CGTase to act upon substrates selected from among starch and decomposition products thereof to produce γ-CD as a main product, the yield is reduced due to hydrolysis of the γ-CD by α-amylase that is present in the crude enzyme. One conceivable method of solving this problem is to purify the crude enzyme and remove the α-amylase. However, in that case, there is a problem of high enzyme production costs. A further method is to inhibit the expression of the amylase gene by an artificial method and relatively increase CGTase activity. However, in this method, it is often difficult to obtain mutant strains in which α-amylase activity alone is selectively inhibited.
It has currently become possible to readily obtain the gene encoding a useful enzyme, create recombinant DNA comprising the gene, and introduce it into a microbe to relatively easily obtain a desired level of enzyme.
Based on this state of the art, the technique of locating the gene encoding γ-CGTase, analyzing the genetic sequence thereof, and improving the productivity and activity of the enzyme by means of transformants into which the gene has been introduced is quite important. Further, once the gene is obtained, mutants can be created to obtain a highly active enzyme. Further, it is anticipated that the techniques of protein engineering can be employed to obtain enzymes with greater heat resistance, pH resistance, and reaction rates.
Accordingly, an object of the present invention is to provide a gene encoding the cyclodextrin glucanotransferase which produces γ-CD as a main product from the substrates selected from among starch and decomposition products thereof, recombinant plasmids comprising this gene and transformants transformed with the recombinant plasmid; a method employing these transformants to manufacture cyclodextrin glucanotransferase acting upon the substrates selected from among starch and decomposition products thereof to producer γ-CD as a main product; and a method employing this recombinant cyclodextrin glucanotransferase to manufacture γ-CD and a CD-comprising composition having a desired CD balance (α-, β-, and γ-CD balance).